Genome mining of cyclodipeptide synthases unravels unusual tRNA-dependent diketopiperazine-terpene biosynthetic machinery

Abstract

Cyclodipeptide synthases (CDPSs) can catalyze the formation of two successive peptide bonds by hijacking aminoacyl-tRNAs from the ribosomal machinery resulting in diketopiperazines (DKPs). Here, three CDPS-containing loci (dmt1-3) are discovered by genome mining and comparative genome analysis of Streptomyces strains. Among them, CDPS DmtB1, encoded by the gene of dmt1 locus, can synthesize cyclo(L-Trp-L-Xaa) (with Xaa being Val, Pro, Leu, Ile, or Ala). Systematic mutagenesis experiments demonstrate the importance of the residues constituting substrate-binding pocket P1 for the incorporation of the second aa-tRNA in DmtB1. Characterization of dmt1-3 unravels that CDPS-dependent machinery is involved in CDPS-synthesized DKP formation followed by tailoring steps of prenylation and cyclization to afford terpenylated DKP compounds drimentines. A phytoene-synthase-like family prenyltransferase (DmtC1) and a membrane terpene cyclase (DmtA1) are required for drimentines biosynthesis. These results set the foundation for further increasing the natural diversity of complex DKP derivatives.

Fig. 1

Chemical structures of compounds 1–13. a Diketopiperazines (DKPs) synthesized by DmtB1 (1–5); b pre-drimentines (pre-DMTs, 6–8 and 13); c drimentines (DMTs, 9–12)

Fig. 2

Comparison of the three CDPS-containing loci dmt1–3. The dmt1–3 are from S. youssoufiensis OUC6819, Streptomyces sp. NRRL F-5123 and S. aidingensis CGMCC 4.5739, respectively

Fig. 3

HPLC traces of culture supernatants of E. coli cells expressing DmtBs

Fig. 4

Mutagenesis of the residues located in the binding pockets of DmtB1. a Superposition of the binding pockets in the DmtB1 model with those in the crystal structure of YvmC. The P1 and P2 of the DmtB1 model are shown in cyan and green, respectively; the P1 and P2 of YvmC (PDB id: 3OQJ) are shown in wheat and brown, respectively. b HPLC traces of the products formation by DmtB1 and its variants (also see Supplementary Fig. 10). c Histogram of the relative formation of various DKPs synthesized by DmtB1 and its variants after 20 hrs of expression in E. coli at 16 °C. The corresponding western blots indicating amounts of proteins are also shown. Error bars represent ± s.d. of three independent experiments. d Total ion chromatogram in multiple reaction monitoring (MRM) mode for compounds 2–5 resulting from in vitro assays of DmtB1 and its variants (L185F and V205M). The relative titers of compounds were compared based on the peak area of MS² fragment 130.11 from each molecular ion

Fig. 5

Heterologous expression of the dmt1–3 loci in S. coelicolor M1146

Fig. 6

HPLC traces of the fermentation products from S. youssoufiensis OUC6819 strains

Fig. 7

Biochemical function of DmtC1. a Schematic representation of DmtC1-catalyzed reactions illustrated by using 2 as substrate. b HPLC traces of DmtC1 biochemical assays

Fig. 8

Impacts of mutagenised DmtA1 on the final cyclization reaction. a Predicted DmtA1 model generated by utilizing TMRPres2D. b HPLC traces of ΔdmtA1 complemented with different dmtA1 variants (also see Supplementary Fig. 59). The cyclization products 9 and 12 were indicated in red. c Relative formation of DMT G (9) production in ΔdmtA1 complemented with different dmtA1 variants in comparison to complementation with wild-type dmtA1 (WT). Error bars represent ± s.d. of three independent experiments

Fig. 9

The CDPS-dependent assembly line of DMT G, 9